In vitro translation is the method producing protein quickly in a reactor without performing cell culture. In the early days of in vitro translation, lower protein productivity was a serious problem. However, it has turned into a very efficient protein production method owing to the optimization of reaction condition and the advanced design of the reactor resulted from the continuous studies by many research groups (Eur. J. Biochem. 239: 881-886, 1996; FEBS Lett. 442: 15-19, 1999).
To increase availability of in vitro translation, it is also required to develop a more efficient and convenient detection method of the cell-free synthesized proteins in addition to the method of improving protein productivity itself. To detect the cell-free synthesized protein easily, a method for selective labeling of the target protein produced from DNA template via translation is required. The selective labeling is very important for the detection because there are already numbers of proteins in the cell-free protein synthesis reaction mixture including enzymes necessary for the production of the target protein. The most widely used method for selective labeling the cell-free synthesized protein is the labeling with radioisotope. In this method, an amino acid labeled with radioisotope is added to the cell-free protein synthesis reaction mixture and then the amino acid is inserted into the growing polypeptide, suggesting that the produced protein is labeled with the radioisotope. At this time, pre-existing proteins in the mixture are not labeled with the radioisotope because those are not the ones that are newly produced, indicating that there is no insertion of the radioisotope into the existing proteins. This conventional method using radioisotope is convenient for labeling the newly produced protein but has a risk of exposing the experimenter on radioactivity. Another disadvantage of this method is inconvenience in the restricted dealing with radioactive materials and radioactive waste.
So, a novel non-radioactive labeling method using fluorophore (fluorescent dye) or biotin, instead of radioisotope, has been developed in order to label and detect the produced protein easily and at the same time in order to solve the problems and inconvenience of the conventional method using radioisotope. The most common non-radioactive labeling method for the selective labeling of cell-free synthesized protein is the method developed by Johnson et al (Biochemistry 15:569-575, 1976). In the method developed by Johnson et al, one amino acid is selected for the labeling of cell-free synthesized protein. Then, an appropriate tRNA corresponding to the selected amino acid is conjugated to the selected amino acid by using aminoacyl-tRNA synthetases. This conjugation reaction between the amino acid and its corresponding tRNA is called aminoacylation. After aminoacylation, fluorophore or biotin for labeling is conjugated to the amino acid constituting the aminoacylated tRNA through functional group on the side chain of the amino acid or α-amino group of initiator methionine constituting the aminoacylated tRNA. As a result, the fluorophore- or biotin-conjugated aminoacylated tRNA was prepared by the above process, which is summarized in FIG. 1. The fluorophore- or biotin-conjugated aminoacylated tRNA used for selective labeling of cell-free synthesized proteins is called “tRNA conjugate for labeling” here in this invention.
The aminoacyl-tRNA synthetases used for aminoacylation have high substrate specificity (these enzymes accept 20 different natural amino acids as a substrate and do not accept non-natural amino acids except only a few). Because of such substrate specificity, the fluorophore- or biotin-conjugated amino acid cannot be conjugated to tRNA by aminoacylation mediated with aminoacyl-tRNA synthetases. Therefore, it is necessary to prepare the aminoacylated tRNA first and later on try to conjugate fluorophore or biotin thereto.
To label the cell-free synthesized protein using “tRNA conjugate for labeling”, the “tRNA conjugate for labeling” is simply added to the in vitro translation reaction mixture. Then, when a protein is generated from DNA, the fluorophore- or biotin-conjugated amino acid is inserted into a growing polypeptide, resulting in the labeling of the cell-free synthesized protein with the fluorophore or biotin.
According to the conventional method developed by E. Johnson, et al, “total tRNA mixture” was used as a tRNA material in the process of preparing the “tRNA conjugate for labeling” for the selective labeling of cell-free synthesized protein. The “total tRNA mixture” indicates the mixture of different types of tRNAs. When the tRNA mixture was used as a tRNA material, following problems were notified. First, when the “total tRNA mixture” was used as a tRNA material, there was a question of efficiency. Despite all different tRNAs provided, it was only one type of tRNA that was actually used for the preparation of “tRNA conjugate for labeling”. That is, only the one that corresponded to the designated amino acid selected by the person who tried to produce the “tRNA conjugate for labeling” was used alone, and no other tRNAs were used at all. That is, there were way many tRNAs remaining unused rather being used. Those unused tRNAs followed the tracks of production of the “tRNA conjugate for labeling” all the time and at last they were included in the final “tRNA conjugate for labeling”. Those tRNAs not corresponding to the selected amino acid could not be conjugated to the selected amino acid, so that they could not be conjugated afterwards to the fluorophore or biotin, either, which means they were useless for the preparation of “tRNA conjugate for labeling”.
When the “tRNA conjugate for labeling” that contains unnecessary impurities, unpaired remaining tRNAs, is used for the labeling of cell-free synthesized protein, the efficiency of protein labeling is accordingly decreased. Besides, signals of those final labeled proteins are not so good, either. That is, signal-to-noise ratio is decreased. This is because fluorophore or biotin is non-specifically bound to the backbone of the unpaired tRNA with generating background fluorescence at the final analysis of labeled protein.
tRNA directly conjugated with fluorophore or biotin through its backbone decreases solubility of the “tRNA conjugate for labeling”, the active ingredient for the protein labeling, indicating that it is difficult to prepare the high concentration of the final “tRNA conjugate for labeling”. To obtain enough signals of labeled protein, a huge amount of “tRNA conjugate for labeling” has to be added. If that is the case, the co-added amount of unnecessary tRNAs is also increased, resulting in more serious background fluorescence.
Considering the above problem, when the conventional method was performed, chromatographic purification was used for the isolation of pure “tRNA conjugate for labeling” after the production of “tRNA conjugate for labeling”. But, this purification of pure “tRNA conjugate for labeling” is very complicated and takes much time.
The present inventors had a belief that the development of a novel preparation method of “tRNA conjugate for labeling” that does not require any additional purification and isolation process was quite necessary and useful in the end. The present inventors were sure that this novel method could be achieved by using only one type of tRNA corresponding to the selected amino acid, instead of “total tRNA mixture”, for the production of “tRNA conjugate for labeling”.
To use only one type of tRNA as a tRNA material for the production of “tRNA conjugate for labeling”, it is theoretically possible to separate only one type of tRNA from total tRNA mixture before aminoacylation. But in reality, it is very difficult to separate one type of tRNA from tRNA mixture because tRNAs are similar to one another in their physical characteristics. So, this could be more difficult and less efficient than the purification and separation of a necessary “tRNA conjugate for labeling” from the final “tRNA conjugate for labeling” preparation.
A more reasonable approach has been made as follows: A gene containing genetic information of a specific tRNA is inserted in cells, and the cells having the gene corresponding to specific tRNA are cultured, resulting in the over-expression of tRNA therein. Then, the cells are harvested and disrupted. Finally, the tRNA is separated from the obtained cell lysate (Proc. Natl. Acad. Sci, USA 84: 334-338, 1987). However, this method also has a problem of contamination of tRNA preparation with unwanted endogenous tRNAs. That is, many types of endogenous tRNAs always exist in the cells at a certain level, indicating the risk of contamination of impurities (different types of endogenous tRNAs) is still there. Even though the ratio of a target tRNA is comparatively higher than that of the case using total tRNA mixture prepared from normal cells, the margin is not so significant and the additional purification is still needed.